The Molecular Characterization Unit (MCU) of the Boston Human Tumor Atlas Research Center (HTA-RC) will comprehensively profile 500 samples (100/yr), collected from melanoma, colon cancer and metastatic breast cancer patients by the Biospecimens Unit (BSU). It will use a suite of complementary, validated cutting-edge cellular/nuclear and spatial technologies to build comprehensive spatiotemporal atlases of the cellular geography of these three cancers. It will also further develop innovative technologies, and perform functional validations of predictions from the Data Analysis Unit (DAU), to understand how dynamic changes in the tumor ecosystem lead to therapeutic resistance. More specifically, the MCU will facilitate the overall mission of the HTA-RC through 4 specific aims. In AIM 1 (the Cellular/Nuclear Branch), the MCU will apply validated single- cell RNA-Seq (scRNA-Seq) and single-nucleus RNA-Seq (snRNA-Seq) methods, in conjunction with bulk whole-exome sequencing and bulk RNA-Seq, to generate genomic profiles of tens to hundreds of thousands of individual cells to develop a comprehensive map of molecularly defined cell types, genetic clones, and states, as well as signatures for spatial studies. In AIM 2 (the Spatial Branch), the MCU will deploy a combination of spatially-resolved cellular RNA (multiplexed error-robust fluorescence in situ hybridization (MERFISH), Spatial Transcriptomics (ST)) and protein profiling (CO Detection of Expression (CODEX), highly multiplex immunofluorescence (HMIF), Multiparameter Ion Beam Imaging (MIBI)) methods to spatially chart cell types, genetic clones, and cell states identified by the cellular/nuclear branch. In AIM 3, we will leverage our team?s expertise to both validate emerging techniques (CITE-Seq) and develop innovative methods to overcome specific challenges?including 3D imaging (whole mount; 3D CODEX), genomic-scale high resolution spatial data (Ex-SEQ and in situ spatial cellular tagging of cells), spatially resolved epigenomics (in situ ATAC-Seq), and proteome-scale measurements in situ (with compressive sensing)?and thereby accelerate the construction of more complete tumor atlases. Finally, in AIM 4, we will test the predictive models generated by the DAU through perturbations and follow-up profiling of banked tumors, cell lines (malignant and TILs), and organoids and organotypic tumor spheroids. The MCU?s established team of scientists?who developed the major methods used in this study?have complementary expertise in single-cell genomics, spatial transcriptomics/proteomics, cancer, immunology, pathology, oncology, technology development, and functional assay. They will work closely with the BSU and DAU to ensure appropriate quality assurance/quality control (QA/QC) measures and sufficient statistical power. The MCU will also be nimble, so as to introduce technologies developed through our collaborative efforts with other Human Tumor Atlas Network (HTAN) Centers, Cancer Moonshot activities, and/or related atlas-building projects. These efforts will be a central part of the goal of understanding how changes in the tumor ecosystem lead to therapeutic resistance in cancer.